Coronavirus Spike Glycoprotein With Improved Expression and Stability

Abstract

Provided herein are mutant coronavirus spike proteins, methods of making and using, vaccines, vectors and nucleic acids, comprising at least one of the following modifications: a short flexible peptide linker or a rigid peptide linker in place of the furin cleavage site loop to genetically link an S1 and S2 subunit; at least one additional disulfide bond; or 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus spike protein has: a higher stability or a higher level of expression when compared to a non-modified coronavirus spike protein. Coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, or an emerging variant thereof. Cunent SARS-CoV-2 variants include, e.g., B.1.1.7, B.1.1.7 with E484K, B.1.135, B.1.351, P.1, B.1.427, D614G, B.1.1351, or B.1.429, Lambda (i.e., C.37), Mu (i.e., B.1.621), Omicron (B.1.1.529) or a variant (including but not limited to BA.1, BA.2, or BA.3) thereof, and others.

1 . A mutant coronavirus spike protein comprising at least one of the following modifications:
 (1) a short flexible peptide linker or a rigid peptide linker in place of a furin cleavage site loop to genetically link an S1 and S2 subunit;   (2) at least one additional disulfide bond; and   (3) 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus spike protein has at least one of: a higher stability or a higher level of expression when compared to a non-modified coronavirus spike protein.   
     
     
         2 .- 3 . (canceled) 
     
     
         4 . The mutant coronavirus spike protein of  claim 1 , wherein at least one of:
 the 1, 2, 3, 4, or 5 proline mutations are selected from F817P, A892P, A899P, A942P, P986K, K986P, V987P, and P987V;   the furin cleavage site loop is at position 676-690;   the short flexible peptide linker is selected from at least one of: GGS (SEQ ID NO:34), GP (SEQ ID NO:35), GPGP (SEQ ID NO:36), GGSGGS (SEQ ID NO:37), or GGGSGGGS (SEQ ID NO:38);   the at least one additional disulfide bond is selected from F43C-G566C, G413C-P987C, Y707C-T883C, G1035C-V1040C, A701C-Q787C, G667C-L864C, V382C-R983C, and I712C-I816C;   the proline mutations are not K986P and V987P mutations;   the at least one addition disulfide bond links the S2 to S2′ subunit, the S1 to S2 subunit, or the S1 to S2′ subunit; or   the higher stability is selected from: increased temperature stability, increased freeze/thaw stability, or increased lyophilization/resuspension stability; and   optionally:
 further comprising a purification peptide at an amino-terminus, a carboxy-terminus, or both. 
   
     
     
         5 .- 9 . (canceled) 
     
     
         10 . The mutant coronavirus spike protein of  claim 1 , wherein the mutant coronavirus spike protein is selected from SEQ ID NOS:1 to 33, or 39 to 42. 
     
     
         11 . The mutant coronavirus spike protein of  claim 1 , wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, Omicron (B.1.1.529) or a variant (BA.1, BA.2, BA.3, BA4.Beta, BQ.1.1, or XBB.1.1) thereof, or an emerging variant thereof. 
     
     
         12 . (canceled) 
     
     
         13 . A nucleic acid encoding the mutant coronavirus spike protein of any one of claims  1 - 12 . 
     
     
         14 . The nucleic acid of  claim 13 , further comprising a vector. 
     
     
         15 . A cell comprising the mutant coronavirus spike protein of  claim 1  or a nucleic acid that expresses the mutant coronavirus spike protein of  claim 1 . 
     
     
         16 . The cell of  claim 15 , wherein the cell is a human cell. 
     
     
         17 . The mutant coronavirus spike protein of  claim 1 , wherein the mutant coronavirus spike protein is formulated into a vaccine. 
     
     
         18 . The mutant coronavirus spike protein of  claim 17 , further comprising an adjuvant, is formulated into a nanoparticle, or is formed into dimers, trimers, or multimers. 
     
     
         19 .- 22 . (canceled) 
     
     
         23 . A method of making a mutant coronavirus spike protein comprising:
 obtaining a nucleic acid sequence encoding a coronavirus spike protein; and   modifying the nucleic acid sequence of the coronavirus spike protein such that the amino acid sequence expressed by the nucleic acid sequence comprises at least one of:
 linking the S1/S2 subunits of a coronavirus spike protein, by deleting a furin cleavage site loop and adding a short flexible peptide linker or a rigid peptide linker; 
   adding at least one additional disulfide bond; and
 adding 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the expressed mutant coronavirus spike protein has at least one of: a higher stability or level of expression, than a non-modified coronavirus spike protein. 
   
     
     
         24 . The method of  claim 23 , further comprising the step of expressing the mutant coronavirus spike protein in a bacteria, fungi, mammalian cell, avian cell, insect cell, or plant cell. 
     
     
         25 .- 26 . (canceled) 
     
     
         27 . The method of  claim 23 , at least one of:
 the 1, 2, 3, 4, or 5 proline mutations are selected from F817P, A892P, A899P, A942P, P986K, K986P, V987P, and P987V;   the furin cleavage site loop is at position 676-690;   the short flexible peptide linker is selected from at least one of: GGS (SEQ ID NO:34), GP (SEQ ID NO:35), GPGP (SEQ ID NO:36), GGSGGS (SEQ ID NO:37), or GGGSGGGS (SEQ ID NO:38);   the at least one additional disulfide bond is selected from F43C-G566C, G413C-P987C, Y707C-T883C, G1035C-V1040C, A701C-Q787C, G667C-L864C, V382C-R983C, and I712C-I816C;   the proline mutations are not K986P and V987P mutations;   the at least one addition disulfide bond links the S2 to S2′ subunit, the S1 to S2 subunit, or the S1 to S2′ subunit; or   the higher stability is selected from: increased temperature stability, increased freeze/thaw stability, or increased lyophilization/resuspension stability; and   optionally:
 further comprising a purification peptide at an amino-terminus, a carboxy-terminus, or both. 
   
     
     
         28 .- 32 . (canceled) 
     
     
         33 . The method of  claim 23 , wherein the mutant coronavirus spike protein expressed by the modified nucleic acid sequence is selected from SEQ ID NOS:1 to 33, or 39 to 42. 
     
     
         34 . The method of any one of claims  23 - 33 , wherein the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, Omicron (B.1.1.529) or a variant (including but not limited to BA.1, BA.2, BA.3, BA4.Beta, BQ.1.1, or XBB.1.1) thereof, or an emerging variant thereof. 
     
     
         35 . A method of immunizing a subject in need thereof against a coronavirus, the method comprising:
 identifying a subject in need of an immunization; and   administering to the subject to a mutant coronavirus spike protein comprising at least one of the following modifications:   a short flexible peptide linker or a rigid peptide linker in place of a furin cleavage site loop to genetically link an S1 and S2 subunit;   at least one additional disulfide bond; and   1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus spike protein has at least one of: a higher stability or a higher level of expression when compared to a non-modified coronavirus spike protein.   
     
     
         36 .- 39 . (canceled) 
     
     
         40 . The nucleic acid of  claim 13 , further defined as comprising:
 one or more mutations that change an amino acid sequence of a coronavirus spike protein by at least one of:   linking the S1/S2 subunits of a coronavirus spike protein, by deleting a furin cleavage site loop and adding a short flexible peptide linker or a rigid peptide linker;   adding at least one additional disulfide bond; or   adding 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus spike protein has at least one of: higher stability or level of expression, than a non-modified coronavirus spike protein.   
     
     
         41 . (canceled) 
     
     
         42 . A vector comprising a nucleic acid sequence encoding a mutant coronavirus spike protein comprising:
 one or more mutations that change the encoded amino acid sequence by at least one of: linking the S1/S2 subunits of a coronavirus spike protein, by deleting a furin cleavage site loop and adding a short flexible peptide linker or a rigid peptide linker;   adding at least one additional disulfide bond; and   adding 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the mutant coronavirus spike protein has at least one of: higher stability or level of expression, than a non-modified coronavirus spike protein; and   optionally:
 the vector is selected for expression in a bacteria, fungi, mammalian cell, avian cell, insect cell, or plant cell; 
 the vector is in a bacteria, fungi, mammalian cell, avian cell, insect cell, or plant cell; or 
 the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, Omicron (B.1.1.529) or a variant (including but not limited to BA.1, BA.2, BA.3, BA4.Beta, BQ.1.1, or XBB.1.1) thereof, or an emerging variant thereof. 
   
     
     
         43 .- 45 . (canceled)